CN110204597B

CN110204597B - Antibacterial peptide and application thereof

Info

Publication number: CN110204597B
Application number: CN201910457698.2A
Authority: CN
Inventors: 杨愈丰; 谢明峰; 王燕; 杨传辉; 卢荣锐; 郭坚; 彭莉萍; 夏嫱; 李均; 刘振勇
Original assignee: Zhuhai Campus Of Zunyi Medical University
Current assignee: Zhuhai Campus Of Zunyi Medical University
Priority date: 2019-05-29
Filing date: 2019-05-29
Publication date: 2021-06-11
Anticipated expiration: 2039-05-29
Also published as: CN110204597A

Abstract

The application discloses an antibacterial peptide in the technical field of biology, and three antibacterial peptides FL15-1, FL15-2 and FL15-3 are obtained by modifying and designing an antibacterial peptide VL15 as a basis, and are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.

Description

Antibacterial peptide and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to antibacterial peptide and application thereof.

Background

Antimicrobial peptides (AMPs) are small molecular active polypeptides which are generated by genes and are synthesized by ribosomes when organisms defend against external pathogen invasion, and are important components of a congenital defense system in organisms. AMPs, as a biologically active small molecule, generally have activity against bacteria, fungi, viruses or protozoa, etc. AMPs may also be used as drug delivery vehicles, anti-tumor agents, immunomodulators, signaling molecules, and the like.

Antibiotics are used as an important clinical medicine, countless lives have been saved since the application of antibiotics, but in recent years, due to the fact that the problems of drug abuse drug residues, bacterial drug resistance and the like are increasingly serious, more and more countries begin to seek antibiotic substitutes, and antibacterial peptide becomes one of the most potential antibiotic substitutes due to the unique biological activity and the special action mechanism different from the traditional antibiotics, and meanwhile, the application of the antibacterial peptide in novel foods, medicines, skin care products and cosmetic preservatives is more and more extensive, so that the antibacterial peptide has a good development prospect.

The research on antibacterial peptides was first traced to 1975, when swedish scientist g.bomam et al injected escherichia coli in pupae before ancient birch, and then found an alkaline polypeptide substance with antibacterial activity, i.e., antibacterial peptide Ceropins, in their blood lymphocytes. Over 40 years of research, over 3000 antimicrobial peptides have been discovered from animals, plants, bacteria and viruses.

While natural antimicrobial peptides have general advantages, there are certain significant disadvantages. A considerable part of natural antibacterial peptides have low bacteriostatic activity, poor stability and high toxicity, or cause hemolysis of eukaryotic cells and the like. In addition, part of the antibacterial peptides have poor inhibition effect on drug-resistant bacteria and cannot meet the requirements of practical application; the artificial antibacterial peptide obtained by modifying or completely synthesizing the natural antibacterial peptide can greatly improve some or even all of the defects so as to adapt to different application requirements. At present, the artificial antibacterial peptide is also thousands of, for example, the artificial antibacterial peptide VL15, the amino acid sequence of which is VLNRLFDKIRQVIRK, (SEQ ID NO.4) the antibacterial peptide is a linear polypeptide and contains 15 amino acid residues, the molecular weight is 1898.35Da, and the isoelectric point is 11.72, but the antibacterial performance of the antibacterial peptide is analyzed, the antibacterial effect of the antibacterial peptide is found to be poor, and the inventor modifies the antibacterial peptide to obtain the antibacterial peptide with better antibacterial performance.

Disclosure of Invention

The invention provides three antibacterial peptides which are improved based on the antibacterial peptide VL15, have better antibacterial performance and have the effect on drug-resistant bacteria.

An antibacterial peptide, herein designated as antibacterial peptide FL15-1 for distinction, the amino acid sequence of antibacterial peptide FL15-1 is shown in SEQ ID No.1, specifically phenylalanine-leucine-lysine-arginine-leucine-phenylalanine-asparagine-lysine-isoleucine-arginine-glutamine-leucine-isoleucine-arginine-lysine, more specifically:

Phe-Leu-Lys-Arg-Leu-Phe-Asp-Lys-Ile-Arg-Gln-Leu-Ile-Arg-Lys, molecular weight is 1974.49Da, isoelectric point is 11.73;

an antibacterial peptide, herein designated as antibacterial peptide FL15-2, the amino acid sequence of antibacterial peptide FL15-2 is shown in SEQ ID No.2, specifically phenylalanine-leucine-lysine-arginine-leucine-phenylalanine-arginine-lysine-isoleucine-arginine-glutamine-leucine-isoleucine-arginine-lysine, more specifically:

Phe-Leu-Lys-Arg-Leu-Phe-Arg-Lys-Ile-Arg-Gln-Leu-Ile-Arg-Lys, molecular weight is 2015.59Da, isoelectric point is 12.48;

an antibacterial peptide is named as antibacterial peptide FL15-3, and the amino acid sequence of the antibacterial peptide FL15-3 is shown in SEQ ID NO.3, specifically phenylalanine-leucine-lysine-arginine-leucine-phenylalanine-arginine-lysine-isoleucine-arginine-lysine-leucine-isoleucine-arginine-lysine, and more specifically:

Phe-Leu-Lys-Arg-Leu-Phe-Arg-Lys-Ile-Arg-Lys-Leu-Ile-Arg-Lys, molecular weight 2015.63Da, isoelectric point 11.10.

The invention also provides another antibacterial peptide, and the amino acid sequence of the antibacterial peptide has a polypeptide with homology of more than 67% with SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3. The polypeptide comprises a similar sequence obtained by substituting, prolonging or truncating 1-5 amino acids in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, and a compound obtained by the similar sequence.

The preparation method of the antibacterial peptide is a solid phase polypeptide synthesis method, and comprises the following steps:

1) swelling resin: 0.6g of 2-Chlorotrityl Chloride Resin having a degree of substitution of 0.4mmol/g was weighed into a reaction tube, added with DCM (15ml/g) and shaken for 30 min.

2) Grafting with the first amino acid: and (3) filtering the solvent through a sand core, adding 3 times of molar excess Fmoc-L-Leu-OH amino acid, adding 10 times of molar excess DIEA, finally adding a small amount of DMF (dimethyl formamide) for dissolving, and oscillating for 1 h. Wash alternately 6 times with DMF and DCM.

3) Deprotection: 15ml of piperidine DMF solution (15ml/g) was added, after 5min the solvent was removed and 15ml of piperidine DMF solution (15ml/g) was added and shaken for 15 min.

4) And (3) detection: the piperidine solution is pumped out, dozens of particles of resin are taken, washed with ethanol for three times, added with ninhydrin, KCN and phenol solution, and heated for 5min at 105-110 ℃, and the positive reaction is obtained when the color turns dark blue.

5) Washing: DMF (10ml/g) was taken twice, methanol (10ml/g) was taken twice, and DMF (10ml/g) was taken twice.

6) Condensation: and (3) dissolving the protected amino acid Fmoc-L-Leu-OH in triple excess and HBTU in triple excess by using DMF as little as possible, adding the dissolved amino acid Fmoc-L-Leu-OH in a reaction tube, immediately adding NMM in ten-fold excess, and reacting for 30 min.

7) Washing: DMF (10ml/g) was taken once, methanol (10ml/g) was taken twice, and DMF (10ml/g) was taken twice.

8) Repeating the two-to-six steps, and connecting the amino acids in the sequence from right to left.

9) After the last amino acid attachment, deprotection, resin wash as follows: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice, DCM (10ml/g) twice, and suction-dried for 10 min.

10) Cleavage of the polypeptide from the resin: preparing cutting fluid TFA 94.5%, water 2.5%, EDT 2.5% and TIS 1%, putting resin into a flask or a centrifuge tube, shaking at constant temperature for a period of time according to the cutting fluid and resin ratio of 10 ml/g: and (4) 120 min.

11) Drying and washing: drying the lysate with nitrogen as much as possible, separating with diethyl ether, washing with diethyl ether for six times, and volatilizing at normal temperature to obtain crude peptide sequence.

12) Purification of the polypeptide by HPLC:

(1) 200mg of the crude peptide is taken and put into a vessel, dissolved by 2-5ml of 50% acetonitrile water solution and subjected to ultrasonic treatment for 2 min.

(2) The lysate was filtered through a 0.45 μm filter.

(3) And (3) analysis: 3 μ l of the filtered solution was taken and analyzed by analytical grade HPLC for crude product for subsequent preparation. The mobile phase is acetonitrile aqueous solution, the time is 30min, gradient elution is carried out, HPLC is balanced for 5min by using an initial gradient, then sample injection is carried out, the initial gradient is 95 percent of water, the acetonitrile is 5 percent, the end proportion is 5 percent of water, and the acetonitrile is 95 percent.

(4) Preparation: injecting sample by using the filtered solution. Preparative HPLC equilibration for 10min, initial gradient water 95%, acetonitrile 5%, end ladder: 25% of water, 75% of acetonitrile and 40min of gradient time. The sample from the detector is collected.

(5) And (3) identification: the collected samples were sampled for purity and MS identification.

13) And (4) freeze-drying the purified solution to obtain a finished product.

14) Sealing and packaging white powdery polypeptide, and storing at-20 deg.C.

The invention provides an application of the antibacterial peptide in preparing broad-spectrum antibacterial drugs for treating gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria and fungal infection.

The invention also provides an application, namely the application of the antibacterial peptide in preparing an antibacterial agent.

Detailed Description

The following is further detailed by way of specific embodiments:

example 1:

1) the product of the antibacterial peptide FL15-1 is an amino acid sequence in SEQ ID NO.1, and the sequence is as follows: Phe-Leu-Lys-Arg-Leu-Phe-Asp-Lys-Ile-Arg-Gln-Leu-Ile-Arg-Lys.

2) The product of the antibacterial peptide FL15-2 is an amino acid sequence in SEQ ID NO.2, and the sequence is as follows: Phe-Leu-Lys-Arg-Leu-Phe-Arg-Lys-Ile-Arg-Gln-Leu-Ile-Arg-Lys.

3) The product of the antibacterial peptide FL15-3 is an amino acid sequence in SEQ ID NO.3, and the sequence is as follows: Phe-Leu-Lys-Arg-Leu-Phe-Arg-Lys-Ile-Arg-Lys-Leu-Ile-Arg-Lys.

Sequence characteristics: the length is 15, the type is an amino acid sequence, the chain type is a straight chain, and the artificial synthesis is carried out.

The antibacterial peptides FL15-1, FL15-2 and FL15-3 of the embodiment are prepared by performing conventional solid phase synthesis on polypeptides by using an automatic polypeptide synthesizer, and the finally obtained antibacterial peptides FL15-1, FL15-2 and FL15-3 are analyzed by high performance liquid chromatography, wherein the purity of the antibacterial peptides FL15-1, FL15-2 and FL15-3 is more than or equal to 98%.

Determination of Minimum Inhibitory Concentration (MIC) of antibacterial peptides VL15, FL15-1, FL15-2 and FL 15-3:

coli (ATCC8739), Pseudomonas aeruginosa (CMCC10104), Staphylococcus aureus (ATCC6538), Candida albicans (ATCC10231) were cultured in log phase, diluted to 5X 10 with 2X liquid MHB medium, respectively⁵CFU/ml. Sequentially adding 50 mul of the antibacterial peptide mother liquor diluted into gradient into a 96-well plate, adding 50 mul of the diluted bacterial liquor into each well, uniformly mixing, standing and culturing for 16 hours at 37 ℃, measuring the light absorption value at 600nm after shaking, and taking 100 mug/ml ampicillin as a positive control. And (4) judging a result: the wells in which no bacterial growth was detected were taken as the minimum inhibitory concentration. The results are shown in the table.

Table 1: MIC values (. mu.M) of antibacterial peptides VL15, FL15-1, FL15-2 and FL15-3 for respective bacteria

As can be seen from the table above, compared with VL15, the modified antibacterial peptides FL15-1, FL15-2 and FL15-3 have obviously improved antibacterial effects on gram-negative and gram-positive bacteria, wherein the antibacterial peptides have the best inhibitory effect on staphylococcus aureus and have better research and development values.

The hemolytic activity detection of the antibacterial peptides VL15, FL15-1, FL15-2 and FL15-3 of the product of the invention comprises the following steps:

1) collecting fresh rat blood, standing for layering, removing upper layer serum, adding physiological saline, gently blowing red blood cells at the bottom of the tube with a straw, centrifuging at 1000rpm for 5min, carefully sucking the upper layer physiological saline with the straw, and discarding until the supernatant is not red.

2) 2 drops of packed red blood cells were removed from the bottom and resuspended in 2.0ml of isotonic PBS to make a 4% red blood cell suspension.

3) Experimental groups: add 50. mu.l of antimicrobial peptide at various concentrations dissolved in isotonic PBS followed by 50. mu.l of the prepared 4% red blood cell suspension.

4) Positive control: mu.l of 0.2% triton X-100, 50. mu.l of the prepared 4% red blood cell suspension, was added to each well. Negative control: mu.l of isotonic PBS and 50. mu.l of the prepared 4% red blood cell suspension were added to each well.

5) After incubation at 37 ℃ for 1h, the wells were centrifuged at 1000 Xg for 5min, 50. mu.l of the supernatant was pipetted from each well into a 96-well plate, and the A value was measured at 415nm to calculate the percent hemolysis of [ (A)_{Sample (I)}-A_{Negative of})/(A_{Positive for}-A_{Negative of})]×100。

The results show that: at a concentration of 78.05 μ M, the hemolysis rate of antibacterial peptide VL15 on erythrocytes was comparable to the negative control; at the concentration of 63.81 mu M, the hemolysis rate of the antibacterial peptide FL15-1 on erythrocytes is equivalent to that of a negative control; at the concentration of 62.02 mu M, the hemolysis rate of the antibacterial peptide FL15-2 on erythrocytes is equivalent to that of a negative control; the hemolysis rate of the antibacterial peptide FL15-3 to the erythrocytes is equivalent to that of a negative control under the concentration of 496.12 mu M, which shows that the osmotic fragility of the antibacterial peptides VL15, FL15-1, FL15-2 and FL15-3 to the erythrocytes can be ignored.

The antibacterial activity of the antibacterial peptides VL15, FL15-1, FL15-2 and FL15-3 of the product of the invention to clinically separated drug-resistant strains is determined:

the results of the measurement of the inhibitory activity of VL15, FL15-1, FL15-2 and FL15-3 on four methicillin-resistant Staphylococcus aureus strains of different origins by the above MIC value measurement method are shown in Table 2.

Table 2: antibacterial effect of antibacterial peptides VL15, FL15-1, FL15-2 and FL15-3 on methicillin-resistant staphylococcus aureus

As can be seen from the table above, compared with VL15, the modified antibacterial peptides FL15-1, FL15-2 and FL15-3 have obvious antibacterial effects on methicillin-resistant staphylococcus aureus strains from different patient sources, and have excellent values in research and development of antibiotic substitute drugs.

The antibacterial activity of VL15, FL15-1, FL15-2 and FL15-3 on the aquaculture pathogenic bacteria vibrio parahaemolyticus and vibrio harveyi strains at the salt concentration of 0.5% and 1% respectively is respectively determined by adopting the MIC value determination method, and the result is shown in Table 3.

TABLE 3 antibacterial effect of antibacterial peptides VL15, FL15-1, FL15-2 and FL15-3 on Vibrio parahaemolyticus and Vibrio harveyi

As can be seen from the table above, compared with VL15, the modified antibacterial peptides FL15-1, FL15-2 and FL15-3 have obvious antibacterial effects on aquaculture pathogenic bacteria vibrio parahaemolyticus and vibrio harveyi strains, the antibacterial effects are basically not influenced by salt concentration, and the antibacterial peptides have excellent research and development values of antibiotic substitute drugs.

In conclusion, the antibacterial peptide product does not produce hemolytic property, has wide antibacterial spectrum and has relatively high antibacterial effect on gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria and fungi. Therefore, the antibacterial peptides FL15-1, FL15-2 and FL15-3 of the product can be preferably applied to the preparation of anti-infective gram-positive bacteria, gram-negative bacteria, drug-resistant bacteria and fungal disease drugs.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Zunyi medical university Zhuhai school zone

<120> antibacterial peptide and application thereof

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<400> 1

Phe Leu Lys Arg Leu Phe Asp Lys Ile Arg Gln Leu Ile Arg Lys

1 5 10 15

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<400> 2

Phe Leu Lys Arg Leu Phe Arg Lys Ile Arg Gln Leu Ile Arg Lys

1 5 10 15

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence

<400> 3

Phe Leu Lys Arg Leu Phe Arg Lys Ile Arg Lys Leu Ile Arg Lys

1 5 10 15

<210> 4

<211> 15

<212> PRT

<213> Artificial sequence

<400> 4

Val Leu Asn Arg Leu Phe Asp Lys Ile Arg Gln Val Ile Arg Lys

1 5 10 15

Claims

1. An antimicrobial peptide, characterized by: the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO.1, and specifically is phenylalanine-leucine-lysine-arginine-leucine-phenylalanine-asparagine-lysine-isoleucine-arginine-glutamine-leucine-isoleucine-arginine-lysine.

2. An antimicrobial peptide, characterized by: the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO.2, and specifically is phenylalanine-leucine-lysine-arginine-leucine-phenylalanine-arginine-lysine-isoleucine-arginine-glutamine-leucine-isoleucine-arginine-lysine.

3. An antimicrobial peptide, characterized by: the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO.3, and specifically is phenylalanine-leucine-lysine-arginine-leucine-phenylalanine-arginine-lysine-isoleucine-arginine-lysine-leucine-isoleucine-arginine-lysine.

4. Use of the antimicrobial peptide of any one of claims 1 to 3 for the preparation of a broad spectrum antimicrobial medicament for the treatment of gram-positive, gram-negative, drug-resistant and fungal infections.

5. Use of an antimicrobial peptide according to any one of claims 1 to 3 in the preparation of an antimicrobial agent.